Exploring endophyte-mediated resistance response against winter cutworm, Noctua pronuba in cool-season turfgrass systems

Progress report for GW23-248

Project Type: Graduate Student
Funds awarded in 2023: $29,902.00
Projected End Date: 04/01/2025
Grant Recipient: Oregon State University
Region: Western
State: Oregon
Graduate Student:
Principal Investigator:
Dr. Navneet Kaur
Oregon State University
Principal Investigator:
Hannah Rivedal
USDA-ARS National Forage Seed and Cereal Research Unit
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Project Information

Summary:

Exploring Epichloë endophytes is an exciting frontier for insect pest management in cool-season turfgrass systems in Oregon. In this study, we proposed to evaluate endophyte-mediated resistance in commercial cultivars of perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), and fine fescue (Festuca rubra) against an economic insect pest Noctua pronuba (L.) (Lepidoptera: Noctuidae), or winter cutworm, a large yellow underwing moth. The research aimed to measure the status of Epichloë endophytes and the response of insect pests in commercial cultivars. Two no-choice greenhouse assays (trail 1 and 2) were conducted to assess insect survival, weight gain, grass biomass, and feeding damage at multiple time points during the 14-day trial duration. Grass tiller samples were analyzed to confirm Epichloë status and alkaloid gene presence post-feeding. Unfortunately, a significant difference both among expected Epichloë incidence and within cultivars was not detected in trial 1, regardless of the grass host species. Additionally, observed Epichloë infection was not an important factor in estimating observed variability in the dependent variables in trial 1. Trial 2 results indicated that the expected Epichloë infection levels in tall fescue did not significantly affect insect mortality or weight gain but were associated with differences in mean feeding damage at 3 and 12 days. However, the impact of expected Epichloë infection status on feeding damage appeared to be overridden by cultivar-specific traits. In perennial ryegrass, low Epichloë infection unexpectedly correlated with higher insect mortality, although this trend was not consistent across all parameters considered. Similar to tall fescue, variations in grass performance metrics in perennial ryegrass were predominantly attributable to cultivar-specific characteristics rather than Epichloë levels.

Project Objectives:

The research objectives of this study include the following:

  1. To assess winter cutworm survivorship and development time on previously identified cool-season turfgrass cultivars in Oregon State University’s NTEP and A-List trials containing a viable endophyte.
  2. To validate the endophyte-mediated resistance response of promising cultivars to winter cutworms by conducting a series of lab experiments whereby endophytic fungi are removed in known E+ (endophyte -positive) turfgrass cultivars and inoculated in E- (endophyte-negative) turfgrass cultivars.

The educational objectives of this project will focus on the following:

  1. To identify potential marketing strategies for the incorporation of endophyte-positive turfgrass cultivars in grass seed and turfgrass systems.
  2. To conduct an educational workshop to increase the knowledge base within the seed industry regarding the role of beneficial endophytes in plant fitness, how to exploit these endophytes in insect pest management plans, and how to retain endophyte viability throughout the seed supply chain.

 

 

Cooperators

Click linked name(s) to expand/collapse or show everyone's info
  • Nicole Anderson (Researcher)
  • Doug Duerst - Producer
  • Julaluck Intasin (Researcher)
  • Alec Kowalewski (Researcher)

Research

Materials and methods:

This study evaluated host plant resistance in terms of winter cutworm feeding response in no-choice assays using commercial perennial ryegrass and tall fescue cultivars with varying endophyte infection levels. 

  1. Assess winter cutworm survivorship and development time on previously identified cool-season turfgrass cultivars in Oregon State University’s NTEP and A-List trials containing a viable endophyte.
  2. Validate the endophyte-mediated resistance response of promising cultivars to winter cutworms by conducting a series of lab experiments whereby endophytic fungi are removed in known E+ (endophyte-positive) turfgrass cultivars and inoculated in E- (endophyte-negative) turfgrass cultivars.

    Cultivars of tall fescue with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified as outlined in Obj 1. However, these cultivars were unavailable commercially, so the proposed objectives (listed above) were modified to conduct two no-choice trials in the greenhouse conditions using four cultivars of two grass species (tall fescue, perennial ryegrass), two levels of endophyte infection (high, low), and two fungicide treatments (no fungicide, fungicide), along with simultaneous winter cutworm (N. pronuba) feeding (winter cutworm, control).

Procedures:

Two no-choice trials were conducted between August and November 2023 at the West Greenhouses, Oregon State University, Corvallis, OR.

Insect collection and rearing method

Winter cutworm, N. pronuba, eggs were collected from commercial grass seed fields in Oregon, USA (45°11'14.4"N 123°14'59.7"W and 44°33'12.3"N 123°18'02.8"W) from August to September 2023. Eggs were checked for parasitism before the introduction into a laboratory for rearing. Healthy eggs and collections without any prior exposure to insecticides were used in the laboratory colonies. The insect colonies were maintained on a general-purpose lepidopteran diet (Frontier Scientific Inc., Newark, DE) using a method slightly modified (Hervet et al. 2016) under a laboratory condition (20 ± 2 °C; 9:15 (L:D) h photoperiod; 70% humidity). One liter of diet was prepared as per the protocol modified from the recipe provided by Frontier Scientific. 875 ml of DI water was boiled in a beaker on a hotplate with a stirring bar, after which 105 g of dry mix were slowly added to the beaker. A rubber spatula was used to vigorously mix the slurry to prevent burning on the bottom of the beaker. Once the diet was fully mixed, 19 g of agar was added to the mixture, followed by vigorous mixing for five minutes. The diet was then transferred into 1 oz-cups, filling them up to ¼ of their volume. 

Plants and seed sources

Cultivars of tall fescue and perennial ryegrass with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified. Two cultivars of each grass species-endophyte combination (e.g. tall fescue-high endophyte, tall fescue-low endophyte, perennial ryegrass-high endophyte, perennial ryegrass-low endophyte) were used in the experiment.

Seeds of the eight cultivars of both grass species were obtained from five different seed storage and distribution facilities in Oregon during Spring 2023, as detailed in Table 1 . The seeds were stored at 4°C when not in use. These seeds were sown in a 40-cell tray insert (150 cm3 per cell) containing RESiLIENCE® potting mix (comprising 35-45% Canadian sphagnum peat moss, processed softwood bark, perlite, coir, and dolomite) (Sun Gro Horticulture, Agawam, MA). Five seeds per cultivar were sown in each cell at a depth of 1 cm. One week after germination, seedlings were manually thinned to one per cell. The grasses were maintained under a 16:8h L:D photoperiod with supplemental lighting from 1000 W sodium vapor bulbs and watered as needed. Day and night temperatures were set at 25 and 20°C, respectively. Another set of the same seeds was treated with a systemic fungicide (Banner Maxx II) containing propiconazole as the active ingredient (Syngenta, Wilmington, DE) to create fungicide-treated counterparts. The seeds were soaked in the fungicide solution (0.5 g a.i .) for 25 min and air-dried on paper towels overnight. The appropriate fungicide rate for controlling endophytes in seedlings was previously determined. Fungicide-treated seeds were planted and maintained in the greenhouse as described above.   

Insect survivorship and development

Eight to nine weeks after the seeds germinated, five plant plugs of each cultivar-fungicide treatment combination were transferred into a 5-gallon plastic storage tote filled with potting mix. The container bases and lids were drilled with six (size) and two holes (size), respectively, to enhance ventilation. Meshes were glued over the holes on the lid and placed between the base and potting mix to prevent larval escape. The potting mix consisted of a 1:1 ratio of G&B Simples Seed Starter Mix (comprising peat moss, perlite, pumice, washed sand) (Kellogg Garden Products, Carson, CA) and autoclaved kiln-dried sand (Marion Ag Services, Inc., St. Paul, OR).

Five second to third instar winter cutworms (with a total weight ranging from 0.14 – 0.55g for trial 1 and 0.33 – 1.13g for trial 2) were added to each plastic container containing five transferred test plants. Every three days after the insect release, the numbers of observed live and dead larvae were recorded, and the aboveground grass area was photographed. The winter cutworm larvae were allowed to feed on the grasses for 14 days before removing them from the arena. Missing larvae were considered dead, and all live larvae were weighed. Each plant was cut at ground level and weighted before three representative tillers were taken and stored in an Agdia’s mesh sample bag at -20°C for endophyte detection using molecular methods (Schardl et al., 2012; Vivuk et al., 2019) (Table 1). This experiment was repeated twice.

Molecular endophyte determination methods

Total nucleic acids were extracted from frozen plant material using the protocol modified from Dellaporta et al. (1983). The modification involved the use of drill press attached with an Agdia tissue homogenizer (Agdia, Inc., Elkhart, IN) to grind samples in the Agdia’s mesh sample bags. The endophyte infection status, infection rate, and chemotype were determined using a high throughput multiplex PCR method described by Takach et al. (2012). The translation elongation factor 1-alpha (tefA) and the following alkaloid biosynthesis-related genes: peramine synthetase (perA), lolC, dmaW, and idtG were amplified from total DNA. All genes have been widely used for endophyte detection and chemotypic analysis of endophytes worldwide (Schardl et al., 2013). The primers used for the multiplex PCR reaction are listed in Table 2. The PCR reactions with a total volume of 25 µl each contained 5× Green GoTaq reaction buffer, 10 μM of each deoxynucleoside triphosphate (dNTP), 1.0-U GoTaq DNA polymerase (Promega Corp., Madison, WI), and each target-specific primer (10 µM). Amplification conditions were two min of initial denaturation at 94°C, 30 cycles of 15s at 94°C, 30s at 56°C, and one min at 72°C, followed by seven min at 72°C (Takach et al., 2012). PCR products were visualized via gel electrophoresis with a 1.5% agarose gel in 1X Tris-borate-EDTA (TBE) buffer following ethidium bromide staining and UV transillumination. The expected fragment sizes for each amplicon was presented in Table 2. Each chemotype was characterized using banding patterns.

Table 1 seed source and cultivar profiles

Grass Species

Cultivars

Endophyte Level provided by the Breeder

Seed Supplier

Tall Fescue

Sidewinder

High

AMPAC Seed Co.

Tall Fescue

Blacktail

High

Columbia Seeds, LLC

Tall Fescue

Bronson

Low

AMPAC Seed Co.

Tall Fescue

Goliath

Low

AMPAC Seed Co.

Perennial Ryegrass

Furlong (LTP-FCB)

High

Lebanon Seaboard Corp.

Perennial Ryegrass

Tee-Me-Up (BSP-25)

High

Bailey Seed & Grain, LLC

Perennial Ryegrass

Mensa

Low

Ledeboer Seed, LLC

Perennial Ryegrass

Sienna

Low

Ledeboer Seed, LLC

 

 

Table 1. Primers used in the multiplex PCR.

Locus Primer  Primer name Primer sequence gDNA size
(bp)
TefA tef1-­exon1d-­1 GGG TAA GGA CGA AAA GAC TCA 860
tef1-­exon6u-­1 CGG CAG CGA TAA TCA GGA TAG  
PER per T2-­F TCTTCAGGCATCGCAGGAAC 600
per T2-­R TCGGCCACCTCCAGCCTGATG  
LOL lolC-­3a GGTCTAGTATTACGTTGCCAGGG 442
lolC-­5b TCTAAACTTGACGCAGTTCGGC  
EAS dmaW-­F4 GTGTACTTTACTGTGTTCGGCATG 282
dmaW-­6R GTGGAGATACACACTTAAATATGGC  
IDT idtG-­F GAGCTTGAGAAGCTTACGAATCC 113
idtG-­R GGGCAATGGAGCGATTCTCTC  

Representative PCR products were purified with the PCR purification kits and sent for sequencing to generate sequence data of Epichloë strains (wild type or novel) among promising cultivars identified during the insect-feeding bioassays.

 

Research results and discussion:

IntasinJulaluck2024_thesis

See the thesis for detailed findings.

Two no-choice greenhouse assays (trail 1 and 2) were conducted to assess insect survival, weight gain, grass biomass, and feeding damage at multiple time points during the 14-day trial duration. Grass tiller samples were analyzed to confirm Epichloë status and alkaloid gene presence post- feeding. Unfortunately, a significant difference was not detected in either the expected Epichloë incidence or within cultivars in trial 1, regardless of the grass host species. Additionally, observed Epichloë infection was not an important factor in estimating observed variability in the dependent variables in trial 1.  Trial 2 results indicated that the expected Epichloë infection levels in tall fescue did not significantly affect insect mortality or weight gain but were associated with differences in mean feeding damage at 3 and 12 days. However, the impact of expected Epichloë infection status on feeding damage appeared to be overridden by cultivar-specific traits. In perennial ryegrass, low Epichloë infection unexpectedly correlated with higher insect mortality, although this trend was not consistent across all parameters considered. Similar to tall fescue, variations in grass performance metrics in perennial ryegrass were predominantly attributable to cultivar-specific characteristics rather than Epichloë levels.

Participation Summary
3 Producers participating in research

Research Outcomes

Recommendations for sustainable agricultural production and future research:

This study demonstrated that Epichloë infection status in tall fescue did not affect N. pronuba mortality or weight gain, but did impact feeding damage on certain sampling intervals during the trial duration. Variability in tall fescue cultivar insect damage metrics was more attributable to cultivar effects than Epichloë infection levels. In perennial ryegrass, Epichloë infection levels affected insect mortality, with higher mortality observed in grasses with expected low Epichloë infection levels.   However, similar to tall fescue cases, variability in perennial ryegrass insect damage metrics was primarily due to cultivar-specific differences rather than Epichloë levels. Previous studies and literature indicated that cultivar often played a more critical role in determining insect resistance than Epichloë infection levels, which aligned with the findings of this study. Moreover, alkaloid gene analysis revealed that peramine was the most common alkaloid gene present in the infected plant population, but this did not correlate with observed insect performance metrics in the trials, suggesting that no-choice assays may not capture the full insect deterrence potential of peramine-producing Epichloë. An extended period of bioassays could be essential to observe sub-lethal effects of alkaloids on N. pronuba.

The unexpectedly low Epichloë infection levels in many cultivars across both trials emphasize the inconsistency and unreliability of Epichloë infection status in turfgrass seeds throughout the Oregon grass seed supply chain. These findings highlight the intricate relationships between grass cultivars, Epichloë infection levels, and environmental conditions in influencing insect resistance and grass performance metrics. Analytical tools such as HPLC and MS will be essential for characterizing and quantifying alkaloids in grass tillers and within insect bodies, thereby enabling a comprehensive understanding of alkaloid production by grass-Epichloë associations and alkaloid uptake by insects. This deeper understanding is crucial for comprehending how Epichloëmediated insect resistance functions against N. pronuba.

1 Grant received that built upon this project
1 New working collaborations

Education and Outreach

3 Consultations
1 Curricula, factsheets or educational tools
1 Workshop field days

Participation Summary:

3 Farmers participated
1 Ag professionals participated
Education and outreach methods and analyses:

Intasin_ESA-meeting_final

IntasinJulaluck2024_thesis

Graduate student Pear Intasin hosted a table at the 2023 Hyslop field day on May 24, 2023, and interviewed a few growers and grass seed industry representatives about their perceptions on utilizing endophyte-mediated insect resistance in cool-season grass species. Pear also prepared a poster to present at the Annual Ryegrass Meeting in Albany in January 2024, but the event was canceled due to inclement weather and icy roads. 

Other presentations to peers included:

Intasin, P., N. Kaur, N.P. Anderson, A. Kowalewski, A. Willette, and H.M. Riveldal. 2024. Evaluating endophyte-mediated resistance against winter cutworm Noctua pronuba in cool-season turfgrass. APS Pacific Division and Conference on Soilborne Plant Pathogens, March 2023, Corvallis, OR. 

Intasin, P., N. Kaur, N.P. Anderson, A. Kowalewski, A. Willette, and H.M. Riveldal. 2024. Evaluating endophyte-mediated resistance against winter cutworm Noctua pronuba in cool-season turfgrass. Pacific Branch ESA Meeting, April 2024, Big Island, Hawaii.

 

Education and outreach results:

Pear Intasin finished the greenhouse trials during winter and is now wrapping up the molecular analysis. Our team will disseminate the research findings during the 2024 Hyslop field day and the grower meetings in the fall of 2024.  

1 Farmers intend/plan to change their practice(s)
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and should not be construed to represent any official USDA or U.S. Government determination or policy.